Variations in nucleic acid sequences and population polymorphisims are studied in the fields of biotechnology, mutagenesis, human genetics, and cancer genetics. A variation in nucleic acid sequence from what previously existed (wild type DNA) is called a mutation. Mutations can include changes in one, or several base pairs in the DNA sequence. Changes can include additions, deletions, inversions or substitutions.
In the field of genetic epidemiology, it is useful to be able to detect patterns of mutations to be probative of the causes of the mutations. In the field of pediatric genetics, detection of mutations is useful to screen for early diagnosis of rare genetic diseases in newborns. In genetic counseling of prospective parents, detection of mutations in their cells is anticipated to be of significant value. Detection of mutations is also useful in the production of biologically produced pharmaceuticals, such as vaccines or recombinant proteins to ascertain compliance with regulatory standards. The detection of mutations is also useful in toxicological studies to determine if genetic damage has occurred due to specific agents, such as additives in cosmetics or environmental contaminants.
Mutations at a specific gene locus (e.g., point mutations) may give rise to significantly altered cellular behavior. However, in any given sample of DNA which may contain mutant DNA, the fraction of mutated DNA molecules out of the total number of DNA molecules present varies greatly. Thus, many pathological conditions can manifest themselves even if only a small fraction of the DNA is mutated. For example, the ability to detect cancer cells by virtue of a mutation present in a small fraction of cells within a tissue or blood sample can be useful to detect metastasis of the cancer, to use as a signal that the cancer is recurring, or as a screen for the initial appearance of a cancer. Additionally, determining the mutational spectra of, for example, the tumor suppressor gene, p53, in non-tumorous tissue of a tumor bearing organ may lead to identification of the probable cause of an individual's tumor. (Harris, C. C., Science, 262:1980-1981 (Dec. 24, 1993).
A number of methods have been used to detect mutant DNA sequences, including isolation of DNA from cells, cloning and sequencing the cloned product. Several electrophoretic methods have been used to separate mutant DNA from wild type DNA including, for example, denaturing gradient gel electrophoresis (DGGE) (Fischer, S. G., and Lerman, L. S., Proc. Natl. Acad. Sci. USA, 80:1579-1583 (1983); Cariello, N. F., et al., Am J. Hum. Genet., 42:726-734 (1988)). However, these methods are tedious and difficult to use. Further, certain common laboratory practices such as labeling DNA molecules with radioactive phosphorous created, in the course of several days, radiolysis reactions which interfere with these methods. Interfering reaction products are also found to arise due to thermolysis in separation extending for many hours at a temperature over 60.degree. C., photochemical reactions with light from ordinary laboratory fluorescent fixtures, and from chemical reactions which presumably involve active oxygen species present in aqueous solutions.
Thus, a fast and reproducible method which can detect mutant DNA sequences present in a sample, including mutant DNA sequences that occur as a small fraction of DNA molecules relative to the total number of DNA molecules present in a sample, would be very important.